EP2820947A1 - Mammifère transgénique non humain pour produire des anticorps d'immunoglobuline E humaine chimérique - Google Patents

Mammifère transgénique non humain pour produire des anticorps d'immunoglobuline E humaine chimérique Download PDF

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EP2820947A1
EP2820947A1 EP13305964.2A EP13305964A EP2820947A1 EP 2820947 A1 EP2820947 A1 EP 2820947A1 EP 13305964 A EP13305964 A EP 13305964A EP 2820947 A1 EP2820947 A1 EP 2820947A1
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human
transgenic
mammal
specific
cells
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Michel Cogne
Brice Laffleur
Armelle Cuvillier
Marie Bosselut
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B Cell Design
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Priority to EP14761405.1A priority patent/EP3016510B1/fr
Priority to US14/900,586 priority patent/US9872483B2/en
Priority to ES14761405T priority patent/ES2716175T3/es
Priority to CA2915113A priority patent/CA2915113C/fr
Priority to PCT/IB2014/062826 priority patent/WO2015001510A1/fr
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/15—Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/20—Animal model comprising regulated expression system
    • A01K2217/206—Animal model comprising tissue-specific expression system, e.g. tissue specific expression of transgene, of Cre recombinase
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/01—Animal expressing industrially exogenous proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/80—Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites

Definitions

  • the present invention relates to a transgenic non-human mammal comprising human immunoglobulin mu and epsilon heavy-chain constant transgenes C ⁇ and C ⁇ inserted in place of endogenous mu heavy-chain switch sequence S ⁇ , and its use for producing chimeric human immunoglobulin E antibodies specific for an antigen of interest.
  • IgE Human immunoglobulins E
  • B cells that express IgE are so rare, transient and and short-lived in vivo in both human and animals that they are extremely difficult if not almost impossible to detect, identify and extract for studies. For these reasons, the knowledge about B cells that undergo class switch recombination to IgE, their terminal differentiation into IgE-secreting plasma cells and the nature of IgE memory, if any, is very limited and it has never been possible until now to design efficient systems for the production of a large repertoire of human, humanized or chimeric human IgE antibodies.
  • hIgE human IgE
  • the production of recombinant human IgE comprises cloning the variable region from the heavy chain of a mouse monoclonal antibody specific for an antigen of interest and rearranging it with a human immunoglobulin epsilon heavy chain constant gene Cepsilon.
  • This method is long, costly and produces only one human IgE antibody molecule specific for a particular antigen of interest so that the method needs to be reiterated entirely for each new human IgE antibody molecule specific for a particular antigen of interest.
  • Humanized transgenic mouse strains comprising from large fragments to complete human immunoglobulin heavy chain (IgH) and kappa light chain (Ig ⁇ ) loci and inactivated mouse IgH and Ig ⁇ loci have been generated and successfully used for generating high-affinity antibodies against various targets ( XenoMouse®: Green et al., Nature Genetics, 1994, 7, 13-21 ; International PCT Application WO 94/02602 ; Mendez et al., Nature Genetics, 1997, 15, 146-156 ; Hu-MAb-Mouse®: Lonberg et al., Nature, 1994, 368, 856-859 ; International PCT Application WO 92/03918 ; KM MouseTM; Tomizuka et al., Proc.
  • Transgenic mice in which IgM expression is constitutively turned into IgA or IgG expression have been generated and successfully used for producing chimeric human IgG and IgA antibodies specific for various antigens of interest (International PCT Applications WO 2005/047333 and WO 2009/106773 ; Duchez et al., Proc. Natl. Acad. Sci. USA, 2010, 107, 3064-3069 ).
  • IgE + B-cells are so transient and short-lived that it is not possible to construct similar transgenic mice to produce human chimeric IgE antibodies.
  • the inventors have made a technological breakthrough by designing the first transgenic animals that can be used for generating hybridomas producing high amounts of high affinity chimeric human IgE antibodies of defined specificity, very easily and quickly.
  • the invention relates to a transgenic non-human mammal, which comprises an endogenous immunoglobulin heavy-chain (IgH) locus comprising the replacement of its mu heavy-chain switch sequence S ⁇ by a human transgenic DNA construct comprising respectively from its 5' to its 3'end, at least:
  • IgH immunoglobulin heavy-chain
  • the human heavy-chain constant genes C ⁇ (C mu or Cmu) and C ⁇ (C epsilon or Cepsilon) which are inserted in place of the switch sequence S ⁇ (S mu or Smu), are therefore located between the intronic activator E ⁇ (E mu or Emu) and the endogenous mouse C ⁇ gene ( figure 1 ).
  • the S ⁇ sequence which is deleted in said transgenic mammal comprises at least positions 136442 to positions 140100 (SEQ ID NO: 1) with reference to mouse chromosome 12 sequence Genbank/EMBL accession number AC073553.
  • the human Cmu transgene in its original configuration (before site-specific recombination has occurred) and the human Cepsilon transgene, after site-specific recombination has occurred, are operably linked to the endogenous mouse heavy-chain variable region genes (V, D, J segment genes) and regulatory sequences (promoter, enhancer(s)).
  • transgenic mammals of the invention produce chimeric human IgMs whose heavy chains comprise a human IgM constant region and a variable region from said transgenic mammal, and no endogenous immunoglobulins M.
  • the chimeric human antibodies generated by the transgenic mammal, in response to an antigen, benefit from a completely diversified repertoire since their heavy-chains whose variable domain VH mainly contribute to the formation of the antibody site correspond to the normal repertoire generated by the rearrangements of the VH, D and JH segments of the murine IgH locus.
  • the transgenic mammals of the invention produce high-affinity human-IgM positive (hIgM + ) B cells producing high affinity chimeric human IgMs specific for said antigen.
  • the B-cells of the non-human transgenic mammals switch expression of chimeric human antibodies isotype from IgM to IgE, to produce functional human-IgE positive (hIgE + ) B cells expressing high affinity chimeric human IgE antibodies specific for said antigen.
  • the non-human transgenic mammal according to the invention has the advantage of generating hybridomas producing high-affinity chimeric human monoclonal IgM or IgE antibodies, specific for any antigen of interest, at levels similar to those usually observed with hybridomas.
  • the methods for producing humanized IgE antibodies specific for an antigen of interest that use the transgenic mammal of the invention are thus much more simple, efficient, rapid and economical than the methods of the prior art.
  • this non-human transgenic mammal which produces detectable levels of functional IgE + B cells can also be used as model to study IgE + B cells maturation in vivo or in vitro following site-specific recombinase induced IgM to IgE switch in the non-human transgenic mammal B cells.
  • non-human transgenic mammal which produces detectable levels of functional chimeric human IgE + B expressing high affinity chimeric human IgE antibodies comprising human IgE heavy chain constant region can also be used as model to study IgE antibodies functions by recruiting human effector cells, for example in double transgenic non-human mammals further expressing a human or humanized IgE receptor.
  • said human heavy-chain constant genes Cmu and Cepsilon each comprise human CH1, CH2, CH3 and CH4 exons and human membrane M1 and M2 exons, separated by the corresponding introns.
  • said human heavy-chain constant gene Cmu comprises the sequence SEQ ID NO: 2
  • said human heavy-chain constant gene Cepsilon comprises the sequence SEQ ID NO: 3.
  • the site-specific recombination sequences are loxP sequences (SEQ ID NO: 5) of Cre recombinase.
  • the human transgenic DNA construct further comprises a selection marker gene.
  • the selection marker gene which is capable of selecting mammalian cells having integrated the human transgenic DNA construct in their genome, is advantageously the neomycine resistance gene (SEQ ID NO: 4).
  • the selection marker gene is preferably flanked by site-specific recombination sequences.
  • the selection marker gene is inserted between the second site-specific recombination sequence (situated immediately downstream of the end of the human Cmu gene) and a third site-specific recombination site situated immediately upstream of the start of human Cepsilon gene, wherein the three site-specific recombination sequences are in the same orientation and the third site-specific recombination sequence is capable of site-specific recombination with the first and/or the second one.
  • the site-specific recombination sequences are advantageously loxP sequences (SEQ ID NO: 5) of Cre recombinase.
  • the selection marker gene is used to select the homologous recombinant non-human mammal embryonic stem cells having integrated the transgenic DNA construct in the targeted endogenous IgH locus. After selection of the homologous-recombinant cells, it can be deleted, in vitro or in vivo, using a site-specific recombinase.
  • said human transgenic DNA construct comprises or consists of the sequence SEQ ID NO: 6.
  • said non-human transgenic mammal it is homozygous for said human transgenic DNA construct.
  • said non-human transgenic mammal further comprises a human immunoglobulin light chain transgene, preferably a human immunoglobulin kappa light chain transgene.
  • said non-human transgenic mammal further comprises a human or humanized high affinity IgE receptor (Fc ⁇ RI) transgene.
  • said non-human transgenic mammal is a knock-in transgenic mammal comprising the replacement of its endogenous high affinity IgE receptor alpha-chain ( ⁇ -chain) gene with its human homologue.
  • ⁇ -chain endogenous high affinity IgE receptor alpha-chain
  • said non-human transgenic mammal further comprises a transgene encoding a site-specific recombinase specific for said recombination sequences, preferably a transgene encoding an inducible recombinase, more preferably a Cre recombinase transgene encoding an inducible Cre, such as for example a tamoxifen-inducible Cre recombinase.
  • a transgene encoding a site-specific recombinase specific for said recombination sequences, preferably a transgene encoding an inducible recombinase, more preferably a Cre recombinase transgene encoding an inducible Cre, such as for example a tamoxifen-inducible Cre recombinase.
  • an example of this transgenic mammal is described in Metzger, D. and Chambon, P., Methods, 2001, 24, 71
  • the transgenic mammal is advantageously homozygous for said transgene(s).
  • transgenic mice comprising at least one, preferably at least two, additional transgenes chosen from a human immunoglobulin light chain transgene, a human or humanized high affinity IgE receptor transgene and transgene encoding a recombinase specific for said recombination sequences, as defined above.
  • the transgenic non-human mammal of the invention comprises said human Cmu and Cepsilon transgenes and eventually said additional transgenes in all its somatic and germ cells. Therefore, the transgenes are transmitted to the transgenic non-human mammal progeny.
  • the invention encompasses also the progeny of said transgenic non-human mammal.
  • the invention encompasses transgenic animals obtained from any mammalian species, preferably from laboratory mammals, more preferably from laboratory rodents.
  • transgenic mammal it is a transgenic mouse.
  • the transgenic mammal of the invention is useful for producing chimeric human antibodies of IgM or IgE isotype, specific for an antigen of interest.
  • a subject of the present invention is the use of the transgenic mammal of the invention for producing chimeric human antibodies of IgM or IgE isotype, specific for an antigen of interest.
  • Another subject of the present invention is a method for producing a chimeric human IgM antibody specific for an antigen of interest, comprising at least:
  • Another subject of the present invention is a method for producing a chimeric human IgE antibody specific for an antigen of interest, comprising at least:
  • the antigen which is used in the present invention is any natural, recombinant or synthetic substance which is able to induce a specific immune response, including the production of specific antibodies, when introduced into a non-human transgenic mammal according to the invention.
  • the antigen comprises one or more of a protein, peptide, lipid, sugar, nucleic acid, and/or mineral.
  • Step a) of the methods is performed by immunizing the transgenic non-human mammal with the antigen of interest according to standard protocols which are known in the art.
  • the antibody may be collected from the serum or B-cell(s) harvested from said mammal.
  • the antibody is collected from B-cell(s) which have been harvested from said mammal and further immortalized.
  • the site-specific recombination may be induced in vivo, in the non-human transgenic mammal or in vitro, in the B-cells harvested from said transgenic mammal and, preferably, further immortalized.
  • the induction in vivo is preferably performed using a transgenic mammal comprising a site-specific recombinase gene encoding a recombinase specific for said recombination sequences, more preferably, said recombinase gene is an additional transgene of the transgenic mammal coding for an inducible recombinase, as defined before.
  • the induction in vitro is preferably performed by immortalizing the B-cells harvested from the transgenic mammal and introducing into the immortalized B cells, an expression vector which encodes the site-specific recombinase, the site-specific recombinase itself or the site-specific recombinase inductor, when the transgenic mammal comprises a recombinase transgene, as defined before.
  • the expression vector is advantageously an expression plasmid comprising a recombinase gene encoding a site-specific recombinase specific for said recombination sequences, wherein said recombinase gene is expressible, in vitro, in said B-cells.
  • the recombinase gene is under the control of a ubiquitous promoter, such as the CMV promoter.
  • the site-specific recombinase which is introduced into the cells is advantageously fused to a cell penetrating peptide (CPP) sequence such as for example the sequence of a CPP derived from HIV Tat basic domain.
  • CPP cell penetrating peptide
  • the site-specific recombination is induced in vitro in the B-cells harvested from said transgenic mammal and further immortalized.
  • B-cell immortalization is performed by fusion of the B-cells with a myeloma cell line, a lymphoblasto ⁇ d cell line, lymphoma cells or an heteromyeloma cell line, according to standard hybridoma production techniques.
  • the B-cells are immortalized by fusion with a murine myeloma cell line, more preferably a murine myeloma cell line like the SP2/0 cell line, which does not produce any murine antibody, is immortalized, and possesses the entire secretion machinery necessary for the secretion of immunoglobulins.
  • the immortalized B-cells are screened for specific antibody production using conventional assays like ELISA. After screening, they are usually cloned using standard methods.
  • the antibodies which are secreted by the immortalized B-cells are harvested from the extracellular medium and usually further purified by conventional techniques known to the persons skilled in the art, such as affinity chromatography.
  • the chimeric human IgM or IgE antibodies specific for an antigen of interest which are produced by the methods of the invention are polyclonal or monoclonal antibodies. Preferably, said antibodies are monoclonal antibodies. These antibodies, in particular the IgE antibodies, are useful for diagnosis and therapy of human diseases in particular, allergy and cancer.
  • the chimeric human IgE antibodies specific for an antigen of interest are used as standards and controls in human allergy assays. Alternatively, they are use as therapeutic antibodies for cancer immunotherapy.
  • the subject of the present invention is also an isolated polynucleotide comprising the human transgenic DNA construct as defined above.
  • the isolated polynucleotide either synthetic or recombinant, may be DNA, RNA or combination thereof, either single- and/or double-stranded, preferably double-stranded DNA.
  • the polynucleotide further comprises DNA fragments from the endogenous IgH locus of said mammal comprising the sequences immediately upstream and downstream of its switch sequence S ⁇ , and said DNA fragments being inserted immediately upstream of the first site-specific recombination sequence and immediately downstream of the human Cepsilon gene, respectively of the human transgenic DNA construct as defined above.
  • the DNA fragments are advantageously of about 5 kb.
  • the DNA fragments correspond advantageously to mouse JH/E ⁇ and C ⁇ regions, respectively.
  • the DNA fragments are from mouse IgH locus. More preferably, the DNA fragments comprise positions 131281 to 136441 (SEQ ID NO: 7) and 140101 to 145032 (SEQ ID NO: 8), respectively of mouse chromosome 12 sequence Genbank/EMBL accession number AC073553.
  • the polynucleotide comprises or consists of the sequence SEQ ID NO: 9.
  • Another subject of the present invention is a targeting vector comprising the human transgenic DNA construct flanked by 5'and 3' homology arms, as defined above.
  • Vectors include usual vectors used in genetic engineering including for example plasmids and viral vectors.
  • the targeting vector is useful for producing the transgenic mammal of the invention.
  • said targeting vector is a plasmid.
  • said targeting vector comprises or consists of SEQ ID NO: 10.
  • the subject of the present invention is also an isolated cell comprising the human transgenic DNA construct as defined above, with the exclusion of human embryonic stem cells.
  • the cell may be eukaryotic or prokaryotic.
  • said cell is a homologous-recombinant cell comprising the human transgenic DNA construct inserted in its endogenous IgH locus, in place of Su.
  • Said homologous-recombinant cell is advantageously obtained by introducing the targeting vector as defined above in a cell, thus allowing the insertion of the transgenic DNA construct in the targeted IgH locus.
  • said cell is an embryonic stem cell from a non-human mammal.
  • the non-human mammal embryonic stem cell is a homologous-recombinant cell as defined above.
  • the homologous recombinant stem cell is useful for producing the non-human transgenic mammal according to the invention.
  • said cell is a B-cell harvested from a non-human transgenic mammal of the invention immunized with an antigen of interest, which B-cell produces a chimeric human antibody of IgM or IgE isotype, specific for an antigen of interest.
  • said B-cell has been further immortalized.
  • Said immortalized B-cells is advantageously an hybridoma.
  • said immortalized B-cell is an IgE producing B-cell further comprising a site-specific recombinase-induced deletion of its human Cmu transgene.
  • the polynucleotide, targeting vector and non-human mammal embryonic stem cell according to the invention are useful for producing the non-human transgenic mammal of the invention.
  • the B-cell harvested from a non-human transgenic mammal of the invention immunized with an antigen of interest, in particular the immortalized B-cell is useful for producing human chimeric IgM and IgE antibodies specific for an antigen of interest.
  • kits for producing chimeric human IgM or IgE antibodies specific for an antigen of interest comprising at least :
  • the different sequences which are used for constructing the polynucleotide and derived targeting vector, cell, transgenic mammals of the invention, including the sequences of human and non-human mammal immunoglobulin genes, are known in the art and accessible in databases.
  • the polynucleotide and targeting vector according to the invention are constructed and introduced in a host cell by the well-known recombinant DNA and genetic engineering techniques using classical methods, according to standard procedures as those described in: Current Protocols in Molecular Biology (Frederick M AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA ) and Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press ) .
  • the polynucleotide is produced by amplification of a nucleic sequence by PCR or RT-PCR, by screening genomic DNA libraries by hybridization with a homologous probe, by total or partial chemical synthesis, or by a combination of said techniques.
  • the insertion of gene fragments into the genome of non-human mammals may be carried out in a random manner, preferably it is carried out in a targeted manner, by homologous recombination with an appropriate targeting vector optionally comprising recombination sequences for a site-specific recombinase such as the loxP sites of Cre recombinase.
  • the inactivation or deletion of gene fragments in the genome of non-human mammals is carried out by homologous recombination with an appropriate targeting vector optionally comprising recombination sequences for a site-specific recombinase such as the loxP sites of Cre recombinase.
  • the transgenic animals according to the invention are obtained by conventional methods for animal transgenesis, according to the standard protocols as described in Transgenic Mouse: Methods and Protocols; Methods in Molecular Biology, Clifton, N.J., Volume 209, October 2002, edited by: Marten H. Hofker, Jan Van Deursen, Marten H. Hofker and Jan Van Deursen, published by Holly T.
  • the multiple transgenic animals are obtained by crossing animals transgenic for human mu-epsilon heavy-chains with one or more animals transgenic for human immunoglobulin light chain, human or humanized high affinity IgE receptor, and/or a site-specific recombinase, as defined above.
  • the chimeric human IgM and IgE antibodies are prepared by conventional techniques known to persons skilled in the art, such as those described in Antibodies: A Laboratory Manual, E. Howell and D. Lane, Cold Spring Harbor Laboratory, 1988 .
  • DNA manipulations were performed using standards protocols such as those described in Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and Son Inc, Library of Congress, USA ).
  • Human mu and epsilon heavy chain constant genes were amplified from human genomic DNA with specific primers containing restriction sites and subcloned into TopoTA 2.1 vectors (INVITROGEN) to yield Topo-hIgM and Topo-hIgM vectors.
  • the vector containing the two arms and the Neo resistance gene (V588) has been described previously ( Duchez et al., Proc. Natl. Acad. Sci. U.S.A., 2010, 107: 3064-3069 and WO 2005/047333 ).
  • Human epsilon heavy chain constant gene was ligated after Xho I restriction of the Topo-hIgE vector and Sal I restriction of the V588 vector.
  • a loxP site has been introduced in 5' of the human mu heavy chain constant gene resulting in a Cla I -Cla I fragment, cloned into the Cla I unique site of the V588+hIgE vector ( Figure 1 ).
  • the resulting targeting vector comprising the human mu and epsilon heavy chain constant genes (human mu-IgH and epsilon-IgH genes; Figure 1 ) has the sequence SEQ ID NO: 10 ( Figure2 ).
  • the human transgenic construct has the sequence SEQ ID NO: 9.
  • the targeting vector has been linearized with PvuI and transfected into E14 ES cells with Amaxa® technology according to the manufactor's instructions.
  • Neomycin 300 ⁇ g/mL was added 24h later to select ES cells.
  • Resistant clones were screened by Southern blot after EcoR I restriction using a mouse Cdelta probe ( Laffleur et al., Methods Mol. Biol. 2012, 901, 149-159 ). Wild type allele results in a fragment of about 8kb and recombinant allele in a band of about 4kb ( Figure 3A ).
  • One clone (114) was used for micro-injection and three agouti positive mice were obtained.
  • mice were screened on blood by flow cytometry with a three colors staining against mouse CD19 (FITC eB1D3), mouse IgM (PE eB121-15F9) and human IgM (APC SA-DA4) clone.
  • Cells were analyzed on a Becton Dickinson Fortessa LSRII. B cells were gated on CD19+ lymphocytes and analysed for human IgM expression.
  • Homozygote mice named hereafter as Mu-Epsilon or InEps mice, expressed only human IgM ( Figure 3B ).
  • Hybridomas were generated from InEps mice according to standard techniques ( Kohler G, Milstein C, Nature, 1975, 256, 495-497 ) and selected for hIgM production ( Figure 4A , left panel) 10 6 hIgM + hybridoma cells were incubated with 2 ⁇ g of a pCDNA3/Cre-recombinase expression vector complexed with 2 ⁇ L of 293fectinTM (INVITROGEN) for 4 h in Opti-MEM® medium (INVITROGEN) at 37 °C in CO 2 atmosphere. The cells were spread over five culture plates (96-well plates, NUNC) in culture medium (DMEM high Glucose/Glutamax/10% Calf Bovine Serum). After 5 days of culture, the hybridoma cells were stained with anti-human IgM APC (SA-DA4) and anti-human IgE FITC (A80-108F).
  • SA-DA4 anti-human IgE FITC
  • Cre recombinase enzyme excises the human mu-IgH transgene and allows the splicing between the rearranged variable genes from the mouse and the human epsilon-IgH transgene, resulting in chimeric human IgE expression ( Figure 4A , right panels).
  • InEps mice were bred with CMV-Cre-ER T transgenic mice ( Metzger D. and Chambon P., Methods, 2001, 24, 71-80 ), a transgenic strain expressing a Cre recombinase fused to a mutated ligand binding domain of the human estrogen receptor (ER), under the control of the ubiquitous cytomegalovirus major IE gene enhancer/promoter, resulting in a tamoxifen-dependent Cre recombinase which is active in a number of cell types, including B cells compartments.
  • the double-transgenic mouse strain homozygous for human Mu-Epsilon IgH genes and at least heterozygous for the CMV-Cre-ER T transgene is called InEps x CreTamox.
  • Splenocytes were isolated from wild-type, InEps and InEps x CreTamox mice according to conventional methods and cultured two days in vitro in presence of LPS (10 ⁇ g/mL) and 4-hydroxytamoxifen (500 nM, SIGMA H7904) in order to activate Cre recombinase activity into InEps x CreTamox mice.
  • LPS 10 ⁇ g/mL
  • 4-hydroxytamoxifen 500 nM, SIGMA H7904
  • Cells were stained with anti-mouse CD19 APC-H7 (1D3), anti-human IgM APC (SA-DA4) and anti-human IgE FITC (A80-108F).
  • Wt cells do not express hIgM or hIgE ( Figure 4B , left panel), InEps mice only express hIgM ( Figure 4B , middle panel). hIgE expression was only detected in IpEps x CreTamox mice in the three independent tests ( Figure 4B , right panels).
  • InEps x CreTamox mice were injected by IP route with tamoxifen (2 mg; SIGMA , ref#T5648). At day four after injection, blood samples were collected from the mice and serum total IgE levels were assayed by ELISA.
  • IgE was detected in the serum at levels of 80.000 U/L ⁇ 20.000 U/L, corresponding to 192 ⁇ 48 ⁇ g/L.
  • Example 2 Production of antigen-specific chimeric human IgE using InEps transgenic mice
  • InEps transgenic mice were immunized by intraperitoneal route with a specific protein antigen from a human pathogen (50 ⁇ g/immunization/mouse) (in ratio 1:1 with Freund Adjuvant) four times every two weeks. Sera were sampled before immunization and 2 weeks after each immunization and stored at -20°C until the ELISA was performed. Briefly, 96 well plates were coated overnight at 4°C with antigen (1 ⁇ g/mL). Plates were blocked with PBS/BSA 2%. Sera were diluted 100 times in PBS/BSA 0.2% and incubated 2h at 37°C. AP-conjugated secondary Ab (goat anti-human IgM, Beckman Coulter) were used at 1 ⁇ g/mL to detect bound hIgM.
  • a specific protein antigen from a human pathogen (50 ⁇ g/immunization/mouse) (in ratio 1:1 with Freund Adjuvant) four times every two weeks. Sera were sampled
  • Figure 5 shows that transgenic mice immunized with the antigen produce specific chimeric human IgM.
  • Hybridomas producing specific chimeric human monoclonal IgM were generated from the immunized transgenic mice according to standard techniques ( Kohler G, Milstein C, Nature, 1975, 256, 495-497 ) and selected for hIgM production by ELISA (Table I).
  • Table I Validation of human chimeric monoclonal IgM for Diagnostic purpose, as calibrator and positive control OD (B-cell Harvest day) Collected splenocytes Screened hybridoma clone First screening positive clone (by ELISA) Second screening positive clone (by ELISA) Stabilized positive clone Clone validated as calibrator and positive control in diagnostic test on automate Mouse #1 1,7895 46.10 6 cells 1530 clones 5 hybridoma clones 9 clones 5 - Mouse #2 1,721 67.10 cells 1150 clones 9 hybridoma clones 40 clones 2 1 clone Mouse #3 0,6295 23.10 6 cells 960 clones 17 hybridoma clones - 3 2 clones
  • transient expression of the Cre recombinase enzyme excises the human mu-IgH gene and allows the splicing between the rearranged variable genes and the human epsilon-IgH gene.
  • 10 6 cells were incubated with 2 ⁇ g of a pcDNA3/Cre-recombinase expression vector complexed with 2 ⁇ L of 293fectinTM (for 4 h in Opti-MEM® medium at 37 ° C in CO2 atmosphere. The cells were spread over five culture plates (96-well plates, NUNC) in culture medium (DMEM high Glucose/Glutamax/10% Calf Bovine Serum). After 5 days of culture, the culture supernatants of the isolated clones were tested by anti-human IgE ELISA.
  • Clones expressing anti-Ag specific human chimeric IgE were identified, subcloned then amplified in culture for IgE production.
  • the clones produce from 0.1 to 50 ⁇ g/ml of anti-Ag human chimeric IgE in standard culture conditions (T75 flask (75 cm2); DMEM high glucose, 10% FCS).
  • Monoclonal chimeric human IgM and IgE production was analyzed by Western-blot ( Figure 7 ). Proteins were separated on SDS-PAGE gels (4%-15% gradient acrylamide gels, BioRad) under non-reducing conditions and transferred on nitrocellulose membranes.
  • the membranes were blocked with 5% milk in PBS and probed in 3% milk/PBS buffer with the primary mouse anti-human IgM (SADA4 clone, BECKMAN COULTER, 1/2000 diluted) or the primary mouse anti-human IgE (Depsilon 2, BECKMAN COULTER, 1/5000 diluted), revealed with the HRP-conjugated goat anti-mouse IgG1 (BECKMAN COULTER, 1/5000 diluted) and visualized by Peroxidase substrat on colorimetric enzymatic reaction (DAB, SIGMA).
  • DAB Peroxidase substrat on colorimetric enzymatic reaction
  • Figure 7 shows that the hybridomas produce chimeric human IgM before induction of site-specific recombination and human IgE after induction of site-specific recombination.

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US14/900,586 US9872483B2 (en) 2013-07-05 2014-07-03 Transgenic non-human mammal for producing chimeric human immunoglobulin E antibodies
ES14761405T ES2716175T3 (es) 2013-07-05 2014-07-03 Mamífero no humano transgénico para producir anticuerpos de inmunoglobulina E humanos quiméricos
CA2915113A CA2915113C (fr) 2013-07-05 2014-07-03 Mammifere transgenique non humain utilise en vue de la production d'anticorps humains chimeres de type immunoglobuline e
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CN108486125A (zh) * 2018-03-27 2018-09-04 重庆金迈博生物科技有限公司 一种核酸分子及其在制备人源单域抗体中的应用

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WO2018144425A1 (fr) * 2017-01-31 2018-08-09 Vanderbilt University Génération d'anticorps monoclonaux ige spécifiques d'allergènes et d'helminthes humains pour une utilisation diagnostique et thérapeutique

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WO2017067043A1 (fr) * 2015-10-21 2017-04-27 重庆市畜牧科学院 Molécule d'acide nucléique chimère et son application dans la préparation d'un anticorps humanisé
CN108486125A (zh) * 2018-03-27 2018-09-04 重庆金迈博生物科技有限公司 一种核酸分子及其在制备人源单域抗体中的应用
CN108486125B (zh) * 2018-03-27 2024-01-05 重庆金迈博生物科技有限公司 一种核酸分子及其在制备人源单域抗体中的应用

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